Human Papilloma Virus (HPV) belongs to the Papovaviridae family, which includes double-stranded members of Papilloma viruses and polyoma viruses. HPV infects the epithelial surfaces of skin or mucosa, causing a warty growth known as condyloma. More than 100 types of HPV have been identified. In addition to benign warty growths, HPV may also be associated with several types of neoplasms.
Cells infected with HPV may undergo changes in cellular morphology, typified by an apparent clearing of the cytoplasm surrounding the nucleus of the HPV infected cell. Such cellular morphology associated with the HPV infected cells may be observed microscopically and used for diagnosis of HPV. Koilocytosis is the term used to describe the cytopathic effect induced by HPV infection. The observed presence of koilocytes in a cytological preparation of exfoliated epithelial cells from the cervix provides one criterion for categorizing a cytologic diagnosis of Low Grade Squamous Intraepithelial Lesion (LGSIL) according to the Bethesda Classification System. Patients classified with a diagnosis of LGSIL, a pre-neoplastic condition, are generally referred to a gynecologic oncologist for colposcopy.
As with many pathologic viruses, it is believed that HPV inside cells (intracellular HPV) is associated with the development of pre-neoplastic disease, while HPV outside the cells (extracellular HPV) is more closely associated with the normal condition of the cells. Therefore, in a diagnostic procedure, it would be desirable to prepare a sample such that it contains no or a fewer number of extracellular HPV, and to base a diagnostic result on such prepared sample. Thus, the determination of HPV cells will correlate more closely with a diagnosis of LGSIL than with a diagnosis of “Within Normal Limits” (WNL).
Various methods have been employed for detection of HPV. For example, liquid-based assay methods typically involve collection of exfoliated epithelial cells and the surrounding extra cellular milieu, and placing the sample into a medium, such as a detergent. The medium dissolves the sample, which is then analyzed to detect HPV. Because the medium employed in such method typically destroys the morphological integrity of the cells, the dissolved solution is homogeneous in that it does not allow intracellular HPV to be distinguished from extracellular HPV.
A pap smear test, which has application in the detection of early cancer of the uterine cervix, may be used to detect HPV. To perform a pap smear test, a physician collects cells by brushing and/or scraping a skin or mucous membrane in a target area with an instrument. The cells are then smeared onto a glass slide, and are fixed and transported to a laboratory where the slide is stained. The glass slide is then examined under a microscope by a cytotechnologist and/or a pathologist to identify cellular abnormalities. During evaluation, a pathologist may employ a polychrome technique, characterized by staining the nuclear part of the cells, to determine the presence of dysplasia or neoplasia. The pathologist may also apply a counter-stain for viewing the cytoplasm of the cells. Because the sample of the pap smear test may contain debris, blood, mucus, and other obscuring artifacts, the pap smear test may be difficult to evaluate, and may not provide an accurate diagnostic assessment of the collected sample.
Cytology based on the collection of the exfoliated cervical cells into a liquid preservative offers many advantages over the traditional method of smearing the cells directly onto the slide. A slide can be prepared from the cell suspension using a filter transfer technique, as disclosed in U.S. Pat. Nos. 6,572,824, 6,318,190, and 5,772,818, which are expressly incorporated herein by reference. Debris, blood and mucus is greatly reduced by the combined method of liquid collection and filtering to transfer the cells onto a glass slide.
Intracellular HPV can be detected by a slide-based assay known as in-situ hybridization. In this method, a sample is first collected. The sample is then denatured, hybridized, washed, and stained according to appropriate probe specifications. After the sample is appropriately prepared, it is then examined on a slide to detect HPV. Although the in-situ hybridization can be used to detect and verify intracellular HPV, it is often difficult and laborious to perform, and the result may be open to subjective interpretation.
An alternative method for detecting HPV involves the use of the Hybrid Capture® System, marketed by Digene Corporation, located in Gaithersburg, Md. The Hybrid Capture® System is a signal amplification assay utilizing antibody capture and chemiluminescent signal detection. Such an assay requires clinical specimens to be combined with an alkaline solution, which disrupts the virus and releases the target DNA. The target DNA then combines with specific RNA probes to create RNA:DNA hybrids, which are then captured onto a solid phase coated with capture antibodies specific for the RNA:DNA hybrids. The captured RNA:DNA hybrids are then detected with antibodies conjugated to alkaline phosphatase. Because the Hybrid Capture® method does not distinguish released DNA originated in extracellular HPV from released DNA originated in intracellular HPV, such method also does not allow intracellular HPV to be distinguished from extracellular HPV.